1. Field of the Invention
The present invention relates to a method of fractional assay of nucleic acids, proteins and other substances, more specifically to a method of capillary electrophoresis suitable for separation of their components close to each other in properties and to an apparatus therefor.
2. Description of the Prior Art
Traditionally, agarose electrophoresis, polyacrylamide gel electrophoresis and other methods have been widely used for fractional assay of nucleic acids, proteins and other substances. However, these methods are disadvantageous to system automation because the gel used therefor is not reusable and requires much time for preparation.
In recent years, attempts have been made to apply capillary electrophoresis to fractional assay of nucleic acids, proteins and other substances. Particularly, its application to fractional assay of nucleic acids has been reported by B. L. Karger et al. and R. G. Brownlee et al.
As a method of carrier-free capillary electrophoresis, Karger et al. used a fused silica capillary and borate-buffered tris(hydroxymethyl)aminomethane containing 7M urea and 0.1% sodium dodecyl sulfate as the electrophoretic buffer to separate a mixture of DNA fragments digested by restriction enzyme [Journal of Chromatography, 458 (1988), pp. 323-333]. Brownlee et al. used a fused silica capillary and an NaH2P04-Na2B407 buffer containing 4M urea and 20 mM cetyltrimethylammonium bromide to separate a mixture of DNA fragments digested by restriction enzyme [Journal of Chromatography, 458 (1988), pp. 303-312]. In both cases, however, the DNA separation mechanism remains unknown. In addition, the former method is faulty in that a peak of unknown origin may appear, and separation becomes difficult to reproduce due to minor differences in sample pretreatment and injection conditions. The latter method does not offer satisfactory separation.
As a method of capillary electrophoresis using gel as the matrix packed in the capillary, Brownlee et al. used a capillary packed with polyacrylamide gel containing 3% T and 5% C to separate a mixture of DNA fragments digested by restriction enzyme [Journal of Chromatography, 458 (1988), pp. 303-312]. Similarly, Karger et al. separated a mixture of d(A)40-60 oligonucleotides [Pro. Natl. Acad. Sci. USA, 85 (1988), pp. 9660-9663]. However, capillary electrophoresis using polyacrylamide gel does not offer high reproducibility of the formation of polyacrylamide gel in the capillary because of the small inside diameter of the capillary, and the mixture separation reproducibility is also poor. Another method of capillary electrophoresis using gel as the matrix uses agarose gel [Brownlee et al., Journal of Chromatography, 458 (1988), pp. 303-312], but agarose gel is mechanically fragile and flows out during electrophoresis. Moreover, the gel melts and flows out from the capillary because of temperature rise due to heat generation during electrophoresis. For these reasons, when a capillary packed with agarose gel is used for electrophoresis, it is not possible to achieve mixture separation with high reproducibility.
As stated above, in fractional assay of nucleic acids, proteins and other substances, agarose electrophoresis and polyacrylamide gel electrophoresis pose problems of much time requirement for gel preparation and difficulty in automation. Also, when used for fractional assay of nucleic acids and other substances, capillary electrophoresis poses a problem of unsatisfactory separation with poor reproducibility, whether or not it uses gel as the matrix.